Heterologous expression of cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and its application in 2-O-α-D-glucopyranosyl-L-ascorbic acid production

Abstract

BackgroundCyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries.ResultsIn this study, the cgt gene encoding α-CGTase from Paenibacillus macerans was codon-optimized (opt-cgt) and cloned into pET-28a (+) for intracellular expression in E. coli BL21 (DE3). The Opt-CGT was purified by Ni2+-NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75 U·mg− 1. In addition, the enzyme was adopted to transform L-AA into 9.1 g/L of AA-2G. Finally, more economic substrates, including β-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5 g/L of AA-2G were obtained, respectively.ConclusionsN-terminal amino acid is critical to the activity of CGTase suggested by its truncation study. Furthermore, when the Opt-CGT was flanked by His6-tags on the C- and N-terminal, the recovery of purification by Ni2+-NTA resin is appreciably enhanced. α-cyclodextrin was the ideal glycosyl donor for AA-2G production. In addition, the selection of low cost glycosyl donors would make the process of AA-2G production more economically competitive.

Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production

“…When using γ-CD and soluble starch as substrate, the yield of AA-2G is low and the price of γ-CD is also high. The experimental results of the present study are consistent with those of Jiang et al (2018). They used soluble starch as substrate and the achieved AA-2G yield was 61% lower than that of α-CD, and 36% lower than that of β-CD (Jiang et al, 2018).…”

“…The experimental results of the present study are consistent with those of Jiang et al (2018). They used soluble starch as substrate and the achieved AA-2G yield was 61% lower than that of α-CD, and 36% lower than that of β-CD (Jiang et al, 2018). Therefore, in this study, β-CD was selected as the donor substrate to synthesize AA-2G.…”

Fisetin glycosides synthesized by cyclodextrin glycosyltransferase from Paenibacillus sp. RB01: characterization, molecular docking, and antioxidant activity

“…The formation of the fisetin/cyclodextrin inclusion complex was also reported in order to improve the property of fisetin ( Zhang et al, 2015 ). CGTase can be found in various bacterial strains such as Bacillus ( Arce-Vázquez et al, 2016 ; de Araújo Coelho et al, 2016 ; Tao et al, 2019 ; van der Veen et al, 2000 ), Pseudomonas ( Jiang et al, 2018 ), and Thermoanaerobacterium ( Leemhuis, Dijkstra & Dijkhuizen, 2003 ). CGTase was used to synthesize various flavonoid glycosides using various flavonoid acceptors such as hesperidin ( Kometani et al, 1994 ), hesperetin ( González-Alfonso et al, 2018 ), naringin ( Kometani et al, 1996 ), epicatechin ( Aramsangtienchai et al, 2011 ) and resveratrol ( Torres et al, 2011 ).…”

Comprehensive study on transglycosylation of CGTase from various sources

“…Furthermore, protein engineering not only improves the transglycosylation of CGTase, but it also can increase the yield of enzymatic products by employing systematic codon optimization strategy. For instance, the systematic codon optimization strategy improves the heterologous expression of Paenibacillus macerans cgt gene in Escherichia coli [ 58 ], and it has been used to increase production of the genistein diglucoside by α-CGTase, which resulted in 1.5-fold increase in production compared with the native α-CGTase [ 42 ].…”